nih public access blocks and new applications of tissue...

The Expanding World of Tissue Engineering: The BuildingBlocks and New Applications of Tissue Engineered Constructs

Pinar Zorlutuna#,Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital,Harvard Medical School, Cambridge, MA, USA.

Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute ofTechnology, Cambridge, MA, USA.

Nihal Engin Vrana#, andCenter for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital,Harvard Medical School, Cambridge, MA, USA.


Ali Khademhosseini*Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital,Harvard Medical School, Cambridge, MA, USA.


Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, MA, USA.

AbstractThe field of tissue engineering has been growing in the recent years as more products have made itto the market and as new uses for the engineered tissues have emerged, motivating manyresearchers to engage in this multidisciplinary field of research. Engineered tissues are now notonly considered as end products for regenerative medicine, but also have emerged as enablingtechnologies for other fields of research ranging from drug discovery to biorobotics. Thiswidespread use necessitates a variety of methodologies for production of tissue engineeredconstructs. In this review, these methods together with their non-clinical applications will bedescribed. First, we will focus on novel materials used in tissue engineering scaffolds; such asrecombinant proteins and synthetic, self assembling polypeptides. The recent advances in themodular tissue engineering area will be discussed. Then scaffold-free production methods, basedon either cell sheets or cell aggregates will be described. Cell sources used in tissue engineeringand new methods that provide improved control over cell behavior such as pathway engineeringand biomimetic microenvironments for directing cell differentiation will be discussed. Finally, wewill summarize the emerging uses of engineered constructs such as model tissues for drugdiscovery, cancer research and biorobotics applications.

Copyright (c) 2011 IEEE.* Corresponding author: Ali Khademhosseini, Ph.D. Harvard-MIT Division of Health Sciences and Technology Wyss Institute forBiologically Inspired Engineering Department of Medicine, Brigham and Women's Hospital Harvard Medical School PRB, Rm 25265 Landsdowne Street Cambridge, MA 02139, USA Tel: 617-388-9271 [email protected].#These authors contributed equally

NIH Public AccessAuthor ManuscriptIEEE Rev Biomed Eng. Author manuscript; available in PMC 2014 January 01.

Published in final edited form as:IEEE Rev Biomed Eng. 2013 ; 6: 47–62. doi:10.1109/RBME.2012.2233468.

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KeywordsBiorobotics; Cell aggregates; modular tissue engineering; organ models; organ-on-a-chip; pre-vascularization synthetic polypeptides

I.IntroductionOver the last 30 years tissue engineering has resulted in important breakthroughs in theunderstanding of cell-material interactions and the host response to biodegradable materialsand their in vivo integration. There is now a deeper appreciation of the effect of physicalproperties on cellular behavior such as material stiffness, surface roughness and porosity [1,2]. From its early stages as single cell type/porous biomaterial constructs to more multi-functional, multi-cellular biomimetic systems, tissue engineering has also providedimportant insights on how the effects of biomaterials on cellular activities can be harnessedfor clinical aims [3].

The initial aim of tissue engineering was to develop tissue or organ substitutes, which are,limited resources in an aging society with prevalent chronic diseases. Driven by the lack ofdonor tissues and the inability of some tissues such as heart and parts of nervous system toheal themselves, tissue engineering methods for replacement tissues and organs havebecome a venue to overcome such problems. Despite limited success in some complexorgans, the promise of substitute tissues has been fulfilled for some targets. The clinicalsuccesses in skin [4], cartilage [5] and more recently in bladder [6] and trachea [7] havealready shown that tissue engineering can fill a gap in the biomedical field. In addition,developments due to trials in other target organs, such as cardiac tissue, have resulted insystems that might not be suitable as implantable systems but can satisfy the ever growingneeds of biomedical field for complex organ and tissue models. Moreover, novel approachesconstantly arise to improve the current tissue engineering efforts by bringing in thedevelopments in other areas of biotechnology and nanotechnology such as pathwayengineering to control cell differentiation, nanoscale bioactive agent patterning ornoninvasive imaging techniques. Modular approaches, rapid prototyping methods andadvances in stem cell research have also contributed to the increasing versatility of tissueengineered constructs. The interactions of different cell types with their surroundingextracellular matrix (ECM) have been recognized as an important determinant of cellbehavior. Individual components of ECM have been widely used as scaffold materials intissue engineering with considerable success. However, the specific composition of ECM ineach organ has proven to be essential for better outcomes. Together with the discovery ofthe importance of cellular microenvironment on stem cell differentiation, obtainingbiomimetic environments has become an important goal. Development of artificial ECMstructures either based on ECM components or synthetic materials is another area wheretissue engineering provides methods for development of cellular microenvironments. It alsobenefits from advances in protein engineering and synthesis. This review aims to cover newdevelopments in these areas and the outlook of tissue engineering, as an expandinginterdisciplinary field.

II. Enhancing Tissue engineering scaffoldsBiodegradable synthetic polymers have been commonly used in tissue engineeringapplications; however, the most commonly used polymers such as poly-L-lactic acid(PLLA), poly L-lactic-co-glycolic acid (PLGA), poly-caprolactone (PCL), generally lack thenecessary signals for cells to reorganize them to generate functioning tissues [8]. Slowremodeling of the scaffolds and prolonged immune response are general problems with such

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scaffolds [9-11]. Moreover, constructs made of a single synthetic polymer often haveisotropic structures which are in contrast to the hierarchical multicomponent organization oftissues. Recently, novel synthetic materials such as engineered synthetic polypeptides havebeen developed to advance structural complexity of the scaffolds. More conventional, yetactive, areas of natural scaffold production are the development of structures based onnatural polymers by using various combinations of ECM molecules or the use ofdecellularized tissues.

A. Enhancing Base MaterialsNatural polymer based structures—ECM components such as collagen, fibronectin,laminin have been commonly used as components of natural scaffolds. Through thesemolecules, cellular activities such as proliferation, differentiation, migration and secretioncan be modulated. Moreover, the multi-component nature of ECM environment hasprominent effects on cell behavior [12]. For example, the composition of ECM affects howcells interact with it, such as the extent of integrin mediated cell adhesion [13]. Theseinteractions can be imitated by the development of artificial ECM from a mixture of naturalECM components. In order to achieve this, commonly used natural polymers such ashyaluronic acid, collagen and gelatin have been further modified to have additionalfunctionalities such as photocrosslinkable sites [14, 15]. These modifications enablecontrollable crosslinking between different ECM components which can be used to regulatecellular behavior better than single component scaffolds [16]. Aside from proteins andpolysaccharides of mammalian ECM, other natural polymers from different sources such asalginate, chitosan and silk fibroin have also been used for obtaining scaffolds. These basematerials have been used in the development of novel natural polymers for tissueengineering such as recombinant chimeric proteins [17]. For example, silk fibroin has beenfunctionalized for antimicrobial activity, conjugated or have been genetically modified tohave additional mechanical properties (such as silk-elastin like protein polymers) [18-20].The versatility offered by recombinant proteins enables more biomimetic scaffolds [21].

One of the advantages of having artificial ECM system is to obtain highly controllable andpreferably reversible 3D systems that would provide an environment very similar to that ofnatural ECM. For example, recently a mixture of collagen and alginate was developed wherethe reversible gelation and dissolution of alginate can be controlled by the addition of ions toregulate the dynamics of cellular spreading and migration [22] (Figure 1). Designed artificialECMs are advantageous as they allow for precise control over composition of the ECM thecells will encounter. For mechanical reinforcement of such ECM-like structures, which aremostly hydrogels, additional materials can be added such as carbon nanotubes [23]. Anotherpossible approach is to control the alignment of fibrillar ECM molecules such as collagen byusing magnetic fields or electrochemical processes. These structures better mimic the tissueswith highly oriented ECM structures [24, 25].

Synthetic polymer based structures—Synthetic biodegradable polymers can bedeveloped with a high level of control as many of their properties, such as molecular weight,viscosity and degradability can be easily controlled by changing synthesis parameters. Thisversatility has resulted in widespread use of several classes of biodegradable syntheticpolymers such as aliphatic polyesters, polycarbonates and polyphosphoesters in biomedicalfields [26]. However, as mentioned before, the interactions of synthetic polymers with cellsare generally indirect and limited. To improve cell response, several approaches areavailable such as blending with natural polymers [27], addition of cell responsive segmentsto the polymer backbone [28] and chemical functionalization of the polymer chains withbioactive agents [29].

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The addition of biological signals to the synthetic materials is a useful tool to control cellularbehaviour [30]. By such modifications, existing biomaterials can be rendered remodellableby cells. Poly(ethylene glycol) (PEG) has been widely used for such systems [31]. Forexample, protease sensitive crosslinkers have been used to generate PEG hydrogels, whichimproved cell proliferation and spreading due to the enzymatic degradation of the hydrogelsby secreted enzymes [32]. Also, the addition of RGD sequences and VEGF directly intoPEG hydrogels has promoted cell attachment and formation of tubules by endothelial cells[33]. Angiogenic potential of growth factor modified PEG hydrogels has also been shown invivo [34].

Another important consideration for engineered scaffolds is mechanical properties. To havemore precise control over scaffolds’ mechanical compatibility with tissues, development ofnew synthetic biomaterials with better defined mechanical, surface and biodegradationproperties is still important. One recent addition to such biocompatible and biodegradablematerials is poly(glycerol sebacate) (PGS). This elastomer, with its highly tailorablemechanical properties, is especially suitable for soft tissue engineering applications, such ascardiac tissue and cartilage [35, 36]. Its mechanical properties have been used to direct thedifferentiation of bone mononuclear cells to smooth muscle cells rather than osteochondralcells by producing a suitable biomechanical microenvironment [37]. Also an improvedsecretion of elastin has been observed in porous PGS scaffolds compared to PLGA scaffolds[38]. Moreover, it can be manufactured in a variety of structures such as electrospunnetworks [39] or accordion-like scaffolds based on honeycomb architecture [40], which canprecisely mimic the anisotropic mechanical properties of soft tissues.

Synthetic polymers can also be produced from nature's building blocks, such as amino acids.The fragile nature of natural polymers generally limits the possible techniques that can beused for their processing. Synthetic polypeptides with functional sequences can overcomethis problem. By designing a polypeptide structure, structural or functional drawbacks of theoriginal ECM molecule such as high temperature sensitivity, high immunogenicity or highenzymatic degradability can be avoided.

As the function and structure of crucial protein fragments are better understood, designedpolypeptides have become a feasible approach to obtain complex tissue engineering scaffoldbase materials. The sequencing and the synthesis of polypeptides became easier and some ofthe polypeptides are already commercially available such as Puramatrix™(RAD16I) [41].Synthetic polypeptide based scaffolds can be formed from designed self-assemblingsequences based on naturally occurring proteins or novel sequences with multiplefunctionalities [41]. The discovery of Arg-Gly-Asp (RGD), as well as other sequences suchas repeat sequences of elastin has shown that full proteins are not always necessary forfunctional outcomes. Today, many synthetic polypeptides such as antimicrobial sequenceswithin some widespread proteins like ubiquitin [42] are commercially produced. Some ofthese polypeptides can self-assemble into various forms. They can also be rendereddegradable by cellular activities at the same time. A functional peptide sequence can be assmall as three or seven amino acids. Smaller building blocks provide precise control ofarchitecture in the new generation of scaffolds. Even with such small segments it is possibleto obtain self-assembled hydrogels [43]. Different amino acid sequences with an inherentability to self-assemble can be used to form a wide variety of structures such as vesicles,ribbons or fibers. Amphiphilicity is a strong tool in nature, as observed in the case ofphospholipids that can form vesicles, membrane or micelles in aqueous solutions. Peptidesdesigned similarly to phospholipids with a hydrophobic head and a hydrophilic tail can alsoform nanoscale vesicles or tubes which can then assemble into networks. This providesnanoscale control over the final scaffold properties. For a hexamer it is possible to define

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many peptide sequences that can self-assemble up to millimeter scale fibers, each of themconferring different properties (Figure 2) [44].

The self-assembly process either happens via alpha-helices, beta-hairpin forming structuresor beta-sheets. For example, by using ionic complementary properties of peptide sequencesbeta-sheet structures with distinct hydrophobic and hydrophilic surfaces can bemanufactured [41]. Self-assembled peptide scaffolds, especially in the form of nanofibers,have been used in in vitro and in vivo studies, such as repair of brain damage in hamstersafter injection of peptide scaffolds [45]. By using peptide moieties, delivery and growthfactor retention properties can be given to such scaffolds and they have been used in a widearray of applications such as nucleus pulposus regeneration, stimulation of angiogenesis oras composites with hydroxyapatite for induction of osteogenesis [46-49]. The self-assemblyproperties can be used in the modeling of diseased states. It is possible to obtain amyloidfiber-like structures with self-assembling synthetic proteins, which is observed in severaldiseases like Alzheimer or Creutzfeld-Jacobs. However, these scaffolds are still singlecomponent scaffolds which cannot imitate the multi-component nature of ECM. As eachpart of ECM has different ways to direct cell behavior, for whole functionality of the nativeECM multi-component scaffolds are necessary. But, obtaining a structure that has allfunctionalities of ECM by modulating each component can be difficult. One way to achievesuch complexity is by using the original tissues or whole organs as scaffolds through adecellularization process. Also by using modular assembly methods microscale control overscaffold architecture is possible.

B. Enhancing ArchitectureBiomimicry has been one of the foci of tissue engineering research and it is recognized as animportant factor for the functionality of an engineered tissue. Many tissues in the body havemodular architectures [50]. Therefore, tissue engineers have been seeking ways to createmodular structures, which can be brought together through random-assembly, self-assemblyor directed-assembly. By this way, a macroscopic construct that possesses microscopicfeatures can be built. Other than being biomimetic, a modular approach can be used topropagate the engineered tissue construct in a predetermined manner, making the modularapproach a high throughput and modular fabrication method.

Random-Assembly—Modularity in the context of tissue engineering can be at differentlevels. As covered in the synthetic polypeptide section, the materials that are used forfabricating the scaffolds can be modular as well as the scaffold itself. The motivation forboth approaches is the same: mimicking the native tissue structure, which is modular in bothmacromolecular (i.e. ECM) and cellular (i.e. liver lobules) level. For example, Davis et al.used genetically engineered protein-based polymers which have regular repeats of lysine andglutamine that can be used for enzymatic crosslinking and hydrogel formation [51]. Thecontrollable nature of the repeating units (i.e. the modularity of its monomers) providedtailorable mechanical and physical properties to the polymer and eventually to the gelproduced. In a more complex manner, multiple hyaluronic acid derivatives were used as“puzzle pieces” to produce tunable composite hydrogels through exploiting the modularityof the polymers comprising it [52]. The material commercialized as Extracel™, whichconsists of 4 basic components to fabricate various gels with different properties bychanging the ratio of these building blocks. A similar approach for fabricating compositehydrogels starting from modular polymers was done using PEG-based micron-sizedhydrogels [53]. In this study, PEG derivatives with different properties were crosslinkedaround living HepG2 liver cells to control mechanical properties, degradation and bioactiveagent delivery. Same modular PEG-based system has also been used for encapsulation ofHL-1 cardiomyocytes, which resulted in long-term viability, growth and expression of

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functional cardiac markers as well as activation peak synchronization upon electricalstimulation. Combination of PEG-based polymers and peptide sequences was also used toproduce modular macromolecules to fabricate synthetic ECM-like structures [54]. Thesewere used for introducing bioactive agents such as VEGF and RGD sequences and the gelstructure was able to accommodate HUVECs in a concentration dependent manner.

Modular tissues have also been proposed as a potential way of providing vasculature to theengineered tissues. In one study, McGuigan et al. fabricated tissue parts using liver cell-encapsulated collagen gels seeded with HUVECs on their exterior surface. The constructswere cultured for 2-3 days until the endothelial cells became confluent and then allowed toself-assemble inside a tube to form a network of interconnected channels (Figure 3) [55]. Asimilar approach was used to fabricate constructs of umbilical cord vein smooth muscle cells(UVSMCs) encapsulated in hydrogels prepared using a lactoyl poloxamine derivative mixedwith collagen. These constructs were then seeded with HUVECs to create the vasculature-like interstitial spaces. Presence of UVSMCs in the hydrogels increased the HUVECattachment and proliferation on the surface compared to constructs containing HepG2 cells[56]. Moreover, the presence of UVSMCs significantly affected the HUVEC phenotype asdetermined by their nitric oxide production [57]. It was possible to perfuse fluids through theinterstitial spaces between the constructs confirming the interconnectivity of the pores [58].When implanted, these modular constructs with endothelial cell lining were able to form astable, perfusable chimeric vascular bed [59]. The modular approach is a strong tool for thedevelopment of vascularized tissues and it was also applied to cardiac tissue engineering. Inthis example, cardiomyocytes were encapsulated in collagen type I-Matrigel mixture and thefabricated constructs were seeded with endothelial cells to create a vascularized tissue [60].Later on, these tissue constructs were assembled into functional, macroporous structureswith adequate contractile properties and gene expression. Presence of endothelial cellsresulted in nonthrombogenic properties and enabled continuous whole blood perfusionwithout clogging [61]. Although interconnected, the flow path was torturous due to therandom assembly. This tortuous nature of the flow path was shown to adversely affect theendothelial cell functionality , depending on the flow rate, due to promotion of disturbedflow causing activated endothelial cells [62]. Therefore, using a random assembly approachmight not be the ideal way of achieving vasculature within modular engineered tissues.

Controlled-Assembly—Researchers have developed approaches that can give morecontrol over the architecture of the final construct compared to random assembly. Controlledassembly of the tissue constructs is important for creating structures with higher biomimicrythat comprise defined architectures and can be used to engineer tissues in a bottom-upmanner. Towards this end, tissue constructs fabricated using photocrosslinkable polymerswere assembled by the aid of hydrophobic-hydrophilic interactions [63-67]. Depending onthe initial geometry of the constructs, various secondary structures were fabricated [66]. Forexample, cell-laden PEG-based hydrogels were produced in arrays of micron-sizedpredefined geometries and assembled in a directed manner to form macron-sized engineeredtissues [64]. To include a microvasculature, more intricate than the mere use of spacesbetween randomly assembled constructs, microchannel networks were incorporated inconstructs by using photolithography. Encapsulated cells were viable after the fabricationprocess and media perfusion through the microvasculature was possible. More control overthe assembly process can be achieved by choosing the construct geometries so that it wouldallow them to be assembled in a directed way through a naturally occurring lock and keymechanism that fit the right constructs together [63, 66-68]. Tissue engineering constructsfabricated using controlled-assembly approaches have been shown to accommodate co-culture of different cell types with high cell viability.

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As an alternative approach for controlling assembly, Yamaguchi et al exploited molecularrecognition to achieve photoregulation of the assembly process [69]. In this studypolyacrylamide was modified with an azobenzene moiety, which can interact differentlywith cyclodextrin groups (again added on polyacrylamide) upon photoisomerization.Through exploiting the different affinities of trans-azobenzene and cisazobenzene tocyclodextrin, adhesion and dissociation of the constructs could be controlled viaphotoirradiation. In another study, either cyclodextrin (host gels) or small hydrocarbon-groups (guest gels) were used to modify acrylamide-based gels and used for directing theirassembly through their mutual molecular recognition [70]. It was also possible to create, sortand selectively assemble subpopulations by modifying the size and shape of the gels (Figure4).

In addition to tissue constructs of cell-laden or cell-seeded materials, constructs thatcomprise of only cells have also been created and assembled towards modular tissueengineering. One approach for directed assembly of such structures that comprise only cellsis to use complementary DNA sequences [71]. In order to modify cell surfaces, azide sugars(i.e. N-azidoacetylmannosamine) were added to cell culture media, which did not show anydetectable adverse affects in cellular metabolism, yet could be incorporated to extracellularglycocalyx [72]. It was possible to assemble cells, which are covalently modified usingcomplementary oligonucleotides, in a controllable manner through the ratio of cells with thedifferent oligonucleotides [71]. For example, with a ratio of 1 to 50 it was possible toassemble cell aggregates within rosette geometry; cells with the less commonoligonucleotide were in the middle of more abundant cells. Similar DNA oligonucleotidemodification of the cell surfaces was also achieved through modification of the cell surfaceusing phosphoramidites in a protein and glycan independent manner [73]. In another study,modification of cell surfaces was achieved through liposome fusion and selective delivery ofketone and oxyamine groups to desired subpopulation of cells [74]. Assembly was achievedthrough oxime ligation and was shown to be effective in controlling the formation of cellaggregates and cell layers to form 3D multilayered structures.

As an alternative approach to chemical modification of cell or cell aggregate surfaces, Brat-Leal et al used physical means for the assembly of cell aggregates by incorporating magneticmicroparticles into them [75]. By this way, it was possible to immobilize, translocate andassemble the aggregates and control these processes temporally.

Decellularized tissues as scaffolds—As long as it can be rendered non-immunogenican allogenic or xenogenic tissue contains all the necessary ECM components in the rightarchitecture. Although decellularized tissues have been in use in the field for a long time, therecent mild decellularization processes [76] that preserve the ECM architecture haveimproved their effectiveness. Since, the intricate structure of the native tissue can bepreserved, the differentiation and orientation of the seeded cells can be directed. Whenapplied to whole organs, this also ensures the presence of well-defined vascular networksthat can be filled faster by endothelial cells in vivo. Some common sources of thedecellularized matrices are adipose tissue, porcine small intestine submucosa and skin,pericardium, trachea and heart valves [77-79].

Decellularization is generally achieved by a combination of physical and chemicaltechniques to induce minimal damage to the ECM structures. This can be achieved either byhyper/hypotonic solutions, treatment with acids and bases or detergent treatments. Physicalmethods including freeze-thawing and pressure application have been also used to minimizechemical use. Decellularized tissues have already achieved success in whole organreplacements in esophagus and trachea [7,80]. However, decellularization is often lengthyand also a slightly deteriorating protocol for the ECM. Moreover, incomplete

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decellularization can lead to immunogenicity and adverse side effects [81]. In addition, theinitial degradation of ECM-based structures is fast and results in rapid loss of mechanicalproperties [82].

Alternatively, there have been efforts to use cell culture techniques to obtain newlysynthesized ECM with a higher level of control over its structure [83]. With the help ofbioreactors and biochemical control of cell behavior, a completely autologous tissue withproperties close to that of the native tissue can be developed. This and similar methods willbe covered in the following section.

III. Scaffold Free ApproachesOn the opposite end of the spectrum, cell-based engineered tissues, without any scaffoldingmaterial have been under investigation for their potential use in regenerative medicine. Cellsare physically not robust enough when they are isolated from their ECM and otherneighboring cells. Thus many attempts using cell suspension injections to the site of adeficient tissue (i.e. infarcted myocardium) have resulted in poor viability, low retention andlimited integration with the host tissue [84]. To avoid these drawbacks, sheets or aggregatesof cells that were cultured in vitro for some time prior to their in vivo application have beentried. Aggregation and sheet formation can increase the physical stability of the cellsthrough cell-cell interactions as well as accumulation of secreted ECM molecules by thecells.

To generate cell aggregates many different substrates have been used including agarose [85],PEG [86, 87], polydimethylsiloxane (PDMS) [88] and poly(N-isopropylacrylamide)(PNIPAAm) [89]. Often, these substrates are prepared using soft lithographical approaches[90], through which the material of choice is cast into a template with desired patterns (i.e.sphere, toroid, cube), and then solidified upon crosslinking or polymerization. Aggregationof the seeded cells on these substrates occurs after a certain incubation time, which isspecific for different cell types.

The properties of the substrate used should enable the retrieval of the cellular constructs.Temperature responsive polymers such as PNIPAAm are very suitable for this process asPNIPAAm becomes hydrophilic and swells when the temperature is changed from 37°C toroom temperature. By using this reversible property, aggregates can be retrieved with a highyield from substrate (Figure 5) [89]. PNIPAAm can be either used as a mold or as a coatingmaterial to modify molds that were made out of other polymers (i.e. PDMS). For example,PDMS molds possessing microgrooves have been coated with PNIPAAm through chemicalcrosslinking and used to form tissue fibers [91].

Usually substrates that have been used for cell aggregation have a flat and smooth structure.This can result in additional diffusion constraints in a system that has already limitations dueto bulkiness of cell aggregates. To avoid this, electrospinning techniques have beencombined with micromolding approaches to achieve PCL substrates with a permeablebottom made of a nanofibrous sheet [92]. Using this system, aggregates of HepG2 cells,embryonic stem cells (ESCs), pancreatic cells and cardiomyocytes have been produced. Asan alternative approach, toroid-shaped cell aggregates have been proposed as a possibleremedy for diffusion and/or vascularization limitations. In this approach, rat hepatocyteswere seeded into agarose microwells to form toroid shaped aggregates after 2 days ofincubation [93]. These aggregates spontaneously combined with each other to form tubularor double-lumen structures.

Cell aggregates have also been used for studying cellular phenotype in a 3D environment[94, 95], and for controlling the differentiation of ESCs [86, 87, 96]. For example, primary

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hepatocyte aggregates produced in a size controlled manner using PDMS concave microwellarrays have shown higher albumin secretion and higher enzymatic activity when they wereco-aggregated with hepatic stellate cells compared to single cell aggregates [94]. In anotherstudy, it was shown that primary ventricular cardiac cell aggregates demonstratedphenotypical differences specifically in expression of tissue maturation markers and underhormonal stimuli compared to 2D culture [95].

One of the most exploited aggregate types is ESC aggregates. Since in their natural state,ESCs are in an aggregated configuration, many researchers have tried to mimic this embryo-like structure in vitro, which is named as embryoid body (EB). These structures have beenutilized to achieve effective differentiation of the ESCs to the desired cell types [97].Although there are other means of creating ESC aggregates such as hanging drop orsuspension culture method, using microwells for aggregation purposes is beneficial sincethis method enables production of more uniform and size-controllable aggregates [97, 98].Especially the size of the EBs is important since it has been shown that the aggregate sizecan influence their differentiation [87]. A number of materials including PEG [86] andhyaluronic acid [99] have been used to fabricate microwells to produce ESC aggregates orEBs. Moeller et al. have examined the effect of various PEG types on aggregation andattachment of ESCs, and for their EB production capacity [100]. Soon after, other highthroughput and facile approaches for EB formation were developed using PDMS [101] andpolyesters [102]. Also, recently, it was shown that the incorporation of biomaterials to cellaggregates could manipulate stem cell behavior [103].

Another widely used and successful approach for tissue engineering in the last decade is theuse of cell sheets. Cell sheets provide more control over the architecture of the formed tissuecompared to cell aggregates since it is possible to align the cells in a particular manner usingmolds with predetermined topological cues [104]. For cell sheet production, againPNIPAAm has been widely used. It is possible to produce cell sheets, pattern the constituentcells of the sheet, and also stack the sheets to form thicker tissues [105]. For example, cellsheets were used to create tissue engineered blood vessel with adequate and biomimeticmechanical properties [106]. Towards this end, circumferential orientation of ECM in thenative vessel was replicated using pNIPAAM coated micropatterned PDMS molds (withgrooves and ridges patterns) as templates for production of aligned cell sheets. By thismethod, it was possible to obtain cell sheets of several layers with aligned cells and ECM,which affected the mechanical properties of the engineered tissue rendering it anisotropic asin the natural tissue (i.e. stronger in the direction of the alignment).

In other studies, cardiac tissue engineering was attempted using this cell sheet-basedapproach [107, 108]. For example, cardiomyocyte cell sheets were stacked together to form3D tissue structures, which could improve the myocardial function upon implantationfollowing a myocardial infarction [109]. Although cell sheets gave better in vivo resultscompared to single cell injection, the cell source used to fabricate the cell sheets wasneonatal rat cardiomyocytes, which is not a sustainable cell source for clinical applications.In order to find a clinically more relevant cell source, ESC-derived cardiomyocytes havebeen used [110]. In this process, the ESCs were cultured in suspension culture in thepresence of growth factors and then enriched for cardiomyocytes. Although ESC-derivedcardiomyocytes did not form sheets, co-culturing with cardiac fibroblasts resulted in cellsheets, which were able to beat spontaneously, synchronize and express Connexin 43. Inanother study, ESCs were used for producing cell sheets without prior selection forcardiomyocytes [111]. Cell sheet formation was shown to enhance cardiomyogenicdifferentiation. The individual cell sheets were used for fabricating 3D scaffolds bysandwiching these cell sheets in between porous scaffolds.

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Cell sheet-based approaches were also tried for liver tissue engineering and characterizedboth in vivo and in vitro for their survival, engraftment with the host tissue and functionality[112, 113]. In an attempt to preserve functionality of the hepatocytes for longer periods,hepatocyte sheets were stacked with endothelial sheets [113]. Cell sheet co-cultures resultedin bile canaliculi network development and higher liver specific gene expression comparedto hepatocyte sheets. Co-culture of endothelial and hepatocyte cell sheets preserved albuminsecretion for 28 days while the single culture of hepatocyte cell sheets showed a decrease inalbumin secretion.

Cell sheets of mesenchymal stem cells (MSCs) from different sources such as periodontalligament, bone marrow and umbilical cord have also been produced [114, 115]. In vivostudies using a swine model showed that periodontal ligament stem cell sheets were moreeffective in regeneration of periodontal defects compared to cell suspension application[114]. Cell sheet formation can be further improved by the addition of exogenous factors.For example, it has been shown that ascorbic acid (Vitamin C) enhanced the cell sheetformation by increasing ECM production [114]. Also, incorporation of gelatin microspheresloaded with transforming growth factor β1 (TGF-β1) facilitated chondrogenic differentiationand cell sheet formation.

As an alternative to thermo-responsive polymer based substrates, polyelectrolyte multilayershave been used to produce cell sheets [116]. In this study, myoblast cell sheets were createdon poly-l-(lysine)/ hyaluronic acid multilayers with a fibronectin top layer. Cell sheets werecollected after dissolution of the polyelectrolyte multilayers by addition of divalent ions tothe culture media.

IV. Cell Sources and Controlling Cell BehaviorA. Stem Cells and Induced Pluripotent Cells

One of the most important aspects of an engineered tissue construct is the cell source. As,over time it has been proven that the primary cells cannot be a sufficient cell source forsome target tissues, other cell sources such as ESCs have been proposed. ESCs can beexpanded in culture indefinitely under proper conditions [117, 118]. ESCs have the potentialto differentiate into all the cell types in the body. ESCs have been successfully differentiatedinto many cell types such as cardiomyocytes [119-121], endothelial cells [122], hepatocytes[123-125], pancreatic beta cells [126], osteoblasts [127, 128], and neural cells [129-133].Despite these properties, ESCs have been the focus of many ethical questions and concernsas well as scientific concerns regarding directing their differentiation in a controlled mannerand without tumour formation [134].

A relatively recent development in the area of genetic manipulation of adult cells resulted inthe development of induced pluripotent stem cells (iPSCs). This cell source has most of theadvantageous properties of the ESCs [135], while being patient-specific and lesscontroversial [136-138]. It has been shown that these cells can be differentiated to many celltypes including hepatocytes [139] and cardiomyocytes [140]. Also, there are few studies thatuse iPSCs for tissue engineering purposes in the literature. For example, there is a recentstudy where the cell source used for cardiac tissue engineering was iPSC-derivedcardiomyocytes [141].

Despite initial promising results with iPSCs, there are concerns on issues such as theefficacy of their differentiation [142] and the additional risk of introduction of somaticcoding mutations [143]. For these cells to be routinely used in tissue engineering andregenerative medicine applications, these problems should be addressed [144]. To use these

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cell sources more efficiently, controlled differentiation of stem cells using biomimeticenvironments and pathway engineering are promising approaches.

B. Controlled DifferentiationCell preparation and differentiation are multi-step and time-consuming procedures, while theyield of the target cell type is usually low. This makes off-the-shelf tissue engineeringproducts hard to achieve; especially for multicellular tissues. Therefore, controlleddifferentiation of stem cells using biomaterials has become an actively researched topic inthe tissue engineering field [145].

Differentiation of stem cells using biomaterials has the advantage of providing a biomimeticenvironment, which can potentially increase the efficacy of the differentiation. Otheradvantages include reducing the processing steps in tissue fabrication by combining celldifferentiation and incorporation as well as enabling simultaneous differentiation of multiplecell types in a single platform for engineering complex tissues. Towards this end, a numberof studies have been conducted to control the stem cell differentiation using physical [146]and chemical [147] properties of the biomaterials.

As the substrate physical properties (i.e. elastic modulus) can affect the lineage commitmentof the stem cells [1], many researchers have investigated ways to exploit this phenomena forpotential tissue engineering applications. For example, MSCs were encapsulated in aninjectable hyaluronic acid-tyramine (HA-Tyr) hydrogel system with tunable mechanicalproperties for potential use in cartilage tissue engineering [148]. Hydrogels with lowercrosslinking degrees yielded more chondrogenic differentiation, with enhanced ECMdeposition. On the other hand, higher crosslinking degrees resulted in generation of cellswith fibrous phenotypes that generated fibrocartilage and fibrous tissue. In another study,collagen and fibronectin functionalized polyacrylamide substrates with mechanicalproperties mimicking tendon and bone tissue were used to modulate bone marrow MSCdifferentiation towards these tissues [149]. It was shown that differentiation could bemodulated using the substrate mechanical properties, but it was interdependent tobiochemical cues as well. Differentiation of MSCs towards endothelial cells was alsostudied in 3D using fibrinogen modified with PEG derivatives [150]. MSCs encapsulated invarious PEGylated fibrinogen hydrogels showed alterations in their differentiation towardsendothelial cells in gels with different mechanical properties. Similarly, when nerveprogenitor cells (NPCs) were cultured in hyaluronic acid with tunable mechanical propertiesranging from that of neonatal brain to adult brain, it was possible to change the NPCphenotype from mature neuronal to astroglial [151].

Another recent way to control stem cell differentiation is to incorporate proteins or peptidesequences to the scaffold, preferably in specific patterns to achieve biomimetic structures.For example, insulin-like growth factor binding protein 4 (IGFBP4) was immobilized onpolystyrene substrates using elastin-like polypeptides in order to direct the differentiation ofESCs towards cardiogenic lineage [152]. In another study, differentiation factors sonichedgehog (SHH) and ciliary neurotrophic factor (CNTF) were patterned in 3D throughmodification of hydrogel chemistry using two-photon laser scanning technology (Figure 6)[153]. Barnase-barstar and streptavidin-biotin chemistries were used for simultaneouspatterning of two different growth factors. Patterning of growth factors with high precisionin 3D can be a promising approach for controlling stem cell differentiation.

C. Pathway engineeringPathway engineering is the science of understanding cells’ major pathways, especially themetabolic ones, and manipulating them through genetic modifications [154]. Pathway

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engineering has been exploited on yeast [155], bacteria [156] and plants [157] foroverproduction of desired metabolites for nearly two decades. This generally involves theenhancement of the expression of a key enzyme in a certain pathway usually by the aid oftranscription factors [158]. More elaborate approaches have emerged such as knocking otherpathways simultaneously while enhancing one or altering more than one enzyme in a givenpathway to have exponential effects. Moreover, with the manipulation of correct pathways,cells could be reprogrammed to a desired phenotype [159].

As a result of the growing knowledge on signaling and metabolic pathways in mammaliancells, pathway engineering approaches have recently been extended to mammalian cells[160-162]. It has also been considered for tissue engineering and regenerative medicineapplications [163]. Currently, the studies in mammalian pathway engineering are forutilization of mammalian cells for production of therapeutic proteins [164]. However,reprogramming a cell through pathway engineering can also be used for controlled andsustainable differentiation of stem cells with high yields as well as for directing othercellular behaviors such as expression and/or secretion of a specific protein. By this way cellscan be manipulated utilizing the knowledge on the signaling pathways for chondrogenicdifferentiation of MSCs [165], skin aging [166], fracture healing of the bone [167], liverregeneration [168] or cellular apoptosis [169], in order to provide solutions for engineeringcomplex tissues. For example, human pluripotent stem cells were reprogrammed intomultipotent neural crest cells through activation of the canonical Wnt signaling andsuppression of the Activin A/Nodal pathway simultaneously [170]. It was also possible tocontrol cell migration and homing through engineering the pathways for surface glycans,which could have profound effects in wound healing and tissue regeneration [171].Although they are few in number, such promising studies suggest that pathway engineeringand cellular reprogramming can potentially be used for solving the existing problems intissue engineering and regenerative medicine.

V. Emerging Tissue engineering ApplicationsAside from the demand for organ substitutes, there is also a growing need for relevant tissuemodels of diseases for basic science purposes or pharmaceutical trials. The most prominentof these efforts based on tissue engineering methods are drug models and cancer models.

A. Tissue Models for Drug TestingDespite the developments in other areas, drug discovery and development still constitute thebiggest portion of the biomedical field. However, as the regulations tighten and the cost ofdrug discovery constantly increases, the drug developers are looking for more intelligentways to test drug candidates. There is a great need for biomimetic testing systems and thecurrent developments in tissue engineering provide a means to help drug discovery and toxinscreening via microtissue based diagnostics and testing [172].

Although, high throughput drug testing systems based only on enzymes have been used fortesting cytotoxicity and drug conversion, they cannot demonstrate the whole metaboliceffect of a drug on liver [173]. The incorporation of cells in microfluidic systems hasimproved such drug platforms. However, 3D effects of drug distribution cannot be evaluatedby 2D culture systems. 3D models developed based on cell encapsulated hydrogels would bea better model of drug distribution [174]. For example, an alginate-based hydrogel systemhas been developed for measuring the toxicity of drugs on encapsulated liver cells withresults comparable to LD50 values in vivo [175]. This system was further modified with theaddition of a cancer cell line, thus simultaneously the toxicity and the anticancer drugefficacy could be tested. The comparison of several widely used drugs’ LD50 values in miceand CT50 values in culture has shown that 3D structures provide a better estimate (R2=0.97)

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than 2D cultures (R2=0.85) [175]. This implies that tissue-like 3D models were a bettermethod for assessing drug effects. Such systems are also important for assessment oftoxicity for growing number of new materials and chemicals in biomedical engineeringfield, which cannot be tested effectively and economically with the currently available tests.Another advantage of miniaturized 3D models is the packing of cells in a smaller volumecompared to a 2D surface. This is a better mimic of cell densities in tissues in vivo.Moreover such artificial constructs would also be an answer to the prevailing questionssurrounding animal use in experiments, particularly for drug and cosmetic tests.

Another aspect of host reaction to drugs is the immune response, which can be unpredictablewith standard in vitro and animal models. Utilization of humanized mice has improved thetest outcomes [176], but still cheaper and animal test-free systems would be desirable. Forthis end, microorganoids resembling the lymph nodes have been developed which can bekept alive and exposed to drugs for several weeks [177]. Analysis of cellular immunity bycytokine release can be done simultaneously with monitoring of other cellular events, suchas proliferation and apoptosis, in this system. Another attempt to provide alternatives toanimal testing via tissue engineering was the development of multicell type artificial corneamimics. Such structures could replace Draize test which utilizes rabbit eyes to test theallergic reactions to new chemicals [178].

The complex, multicellular structure of some organs is hard to imitate. Especially theproblems related to the vascularization of engineered construct above volumes of severalmm3 limit organ production at macro scale. However, with the developments inmicrofluidics-based systems, miniaturized organs can be produced. With the help ofmicrofabrication techniques these miniaturized organs can be produced as valuable drugdevelopment tools.

These tissue-on-a-chip or organ on-a-chip structures can be viewed as miniaturized organswhere all the physiological activities pertinent to the metabolization of a drug or a toxin canbe closely mimicked. Microscale tissues are advantageous for monitoring due to decreasedamount of reagent use, smaller sample size and the possibility of high throughput analysis.Moreover, via miniaturization, it is possible to mimic physiological shear stress conditionsbetter as well as to provide relevant physiological fluid composition spatially andtemporally. Analysis of drug candidates with microtissues can both increase the reliability ofthe tests and enable detection of candidates that can be eliminated in animal tests due tofalse negative results.

Even though microengineered tissue based tests would be an important improvement, itcannot answer the questions related to another important aspect of drug testing andmonitoring which are the short and long term systemic effects of the drugs. For example, asingle toxicity test based on an artificial liver system would not account for the possible sideeffects of the metabolic by-products of the hepatocyte activity on the drug on other tissues.A possible solution proposed for this problem is the body-on a chip systems. By connectingseveral organs, it is possible to obtain a body-on-a-chip system where the effect of one drugor therapy on multiple organs can be elaborated. Already, there are several such systemsavailable, such as gastrointestinal tract models, kidney models and lung air sac models [179,180]. In such systems, reciprocal interactions of the cells can be monitored, such as increasein hepatic glucose synthesis by hepatocytes in the presence of endothelial cells [181].

A body-on-a-chip will offer multiple advantages: i) metabolization of the drugs and theirpossible systemic effects can be monitored and the sequence of the events can be elucidated,ii) small scale effects that can be missed in animal tests which can result in negativeoutcomes in later clinical trials can be detected, iii) testing of drug-drug interactions can be

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done in a cheaper and more efficient way for several organs at the same time [182]. Theywill also allow elaboration of the mechanisms of the drug toxicity and side effects withoutanimal studies and in a more complex manner than cell culture studies.

However, having many cell types in a small system creates some practical problems, such asthe composition of the culture medium [183]. Some solutions have already been availablesuch as development of a blood surrogate which would be suitable for all cell types. Otheroptions are the separation of the feeding of each microorgan so that they can be in contactwith their own medium where metabolite exchange between microorgans is achieved byseparate connections or addition of controlled delivery functions into the chip for specificgrowth factor delivery to each compartment [181, 184, 185].

Another important issue in the development of such systems is the utilization of suitablemethodologies for their monitoring. Since the aim is to observe long-term effects and alsotemporal changes in responses, noninvasive methods for visualization of tissue integrationand cellular activity at high resolutions are necessary. The microscopy methods such asLens-free microscopy [186], Coherent Anti-Stokes Raman Microscopy (CARS) andSecondary Harmonic Generation (SHG) [187, 188] can provide real-time information aboutthe model ECM and cells (Figure 7).

As for monitoring of metabolic activity, since in such small volumes collection of analytes isproblematic, general choice of analysis is electrical or optical methods [189], such ascellular impedance measurements via surface plasmon resonance. Also, in addition tomicroscopy methods such as time lapse microscopy, by using fluorogenic molecules specificinteractions of cells with drugs or metabolites can be monitored. For example,autofluorescence of doxorubicin allows monitoring of its uptake [190]. Correlation of cell'sphysical properties with their activities is a strong indicator of their health that can beincorporated into long term monitoring of cellular activities.

Other possible outcomes of tissue engineering would be disease models, such as robust invitro 3D models which can closely mimic the in vivo condition. These models can be usedfor basic science research and also for therapeutic drug testing [191]. Especially with thestrong indications of non-applicability of animal test results for human trials, 3D artificial,diseased human tissues might be a reliable model for drug testing.

B. Cancer ModelsThe ability to develop healthy and diseased tissue at the same time, possibly from the samecell source is a strong tool to understand the onset and propagation of diseases such ascancer. The large variation of cancer cells from patient to patient such as differences in theirmetastasis behavior, requires models that can reflect these discrepancies with high fidelity.

In vitro 2D culture of cancer cells cannot provide information about their movement in 3D.A tissue engineering-based cancer model can reflect the invasive properties of thecarcinomas. It can also be used to monitor the feedback mechanisms between tumor andstromal cells and also the interactions of cancer cells with different cytokines.

For modeling metastasis, the 3D cell migration process needs to be imitated. There are newtechniques available for achieving control over the cell migration and spreading in 3Dstructures. Tumorogenesis can be accurately imitated in systems which contain all relevantcell types together with a biomimetic ECM structure. For example, for breast cancer models,2D culturing of epithelial cells will give an incomplete picture as epithelial-stromainteraction is an important part of tumor progression. The addition of ECM components viaa natural scaffold would also be necessary to reinforce the robustness of the model. In the

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absence of ECM, 3D cancer models have been produced with spheroid cultures, which canbe prepared from epithelial tumors (homotypic aggregation) and can be rendered also multi-cellular (via heterotypic aggregation) to model the interactions of stroma and the cancer cells[192]. Such structures not only can be used to model tumor architecture but also provide allthe necessary autocrine and paracrine effects to better mimic the tumor microenvironment.Mesenchymal induction of basement membrane deposition by epithelial cells [193] has beenshown to be an important determinant in development of epithelium based cancer models.For example, laminin rich ECM was previously used to distinguish the differences betweennormal and malignant breast cells. The healthy cells formed polarized colonies and thecancerous cells grow into proliferative colonies with no definite organization. This modelcan be used to elaborate the differences between healthy and diseased structures which willgive a more complete understanding of how the disease progresses than 2D culture models[194].

Another important factor during embryonic development and cancer metastasis is theepithelial to mesenchyme transition. The interaction of mesenchyme and the epitheliumdefines their function and structure in a reciprocal way during development. Understandingthe underlying principles of this transition via co-cultures in scaffold systems would help inunderstanding the complex interactions between these cell types. For example, it has beenshown that epithelialmesenchyme transition resulted in cells that demonstrate stem cell-likeproperties [195]. Imitation of the microenvironment that leads to this event would bebeneficial both for cancer research and standard tissue engineering applications [196].

These developments in artificial disease models would also help in optimization of surgicalprotocols. Cryosurgery is a growing field in the cancer resection area as it is a minimallyinvasive technique with high efficiency. But the microenvironment induced by theapplication of extremely low temperatures, which is below the thresholds for thermodamage,can cause damage to the area surrounding the target. The optimal application conditions andtheir long term effects will only be available after years of data gathering with conventionalmethods. However tissue engineered constructs can be exposed to the same conditions of thesurgery and the effect of the treatment can be visualized in real–time. This way, in-depthmeasurements of the physical conditions within the tissue can be made and the surgeryconditions can be rectified accordingly [197]. Moreover, these kinds of models can befurther used for the tissue preservation and organ preservation studies and the complexeffect of freezing on the tissues.

C. BioroboticsBiorobotics is a field that aims to design robots that can mimic animal or human movementsand to analyze natural control systems to use them in new robot designs. For example, eachmuscle in animals can act as a multiple actuator with the ability to respond to multitudes ofinputs. This provides a substantial advantage to the single actuator/ single input systems inconventional robots. Bio-inspired “cellular actuators” based on piezoelectric materials havebeen developed to mimic this property [198]. Aside from physical biomimicry, recentlyresearch focus also turned on using cell based systems as actuators. Cell types that can act asactuators such as vorticella [199], cardiomyocytes, myoblasts can be used to develop MEMSsystems that can also function in aqueous conditions. For a feasible actuator high density ofcells is necessary. For this end, there have been trials with extracted tissues such as dorsalvessel obtained from insects [200] or in vitro assembled systems based on fibroblasts andmyoblasts that can actuate microdevices [201]. But better actuators with more control overcell localization can be obtained by tissue engineering methods. For example, myoblastslabeled with magnetite cationic liposomes have been used to form an artificial muscleconstruct under magnetic force to act as a bioactuator [202]. Also cardiomyocytes withinECM-based gels has been developed as microdevice actuators [203]. There are also ongoing

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trials to use neuron cell cultures as biological processors that can control robotic systems[204], which can benefit from tissue engineering methodologies. Tissue engineeredactuators would provide more sophisticated structures with more control over mode ofactuation. They can also be used to form the interface between robotic systems and tissues.Although, most of the tissue engineering efforts are limited to mammalian cells forregenerative medicine purposes, another possibility in biorobotic/tissue engineeringinterface is the utilization of cold-blooded species’ cells such as insects and zebrafish, whichhave less stringent requirements for culture.

VI. future perspectivesThe ties and contribution of tissue engineering methodologies to biotechnology have beengrowing strongly. With recent clinical successes, combined with the better understanding ofhost/biomaterial interactions tissue engineering still has room to grow. The novel scaffoldsare smart in many ways, but they generally lack the temporal control over the modelingprocess in vivo. The next generation would also incorporate structures that would activelymeasure the health of the implant. Such lab on a chip applications in vivo, could improve thequality of monitoring available for implant systems [205]. Also developments in diseasemodels and body-on-a-chip applications and demands in biorobotics will create newrequirements for the next generation of tissue engineered constructs which will necessitatemore widespread use of new methodologies such as pathway engineering or use ofalternative cell sources.

AcknowledgmentsThis paper was supported by the Institute for Soldier Nanotechnology, National Institutes of Health (HL092836,EB02597, AR057837, HL099073), the National Science Foundation (DMR0847287), and the Office of NavalResearch Young Investigator award

References1. Engler AJ, et al. Matrix Elasticity Directs Stem Cell Lineage Specification. Cell. Aug.2006

126:677–689. [PubMed: 16923388]

2. O'Brien FJ, et al. The effect of pore size on cell adhesion in collagen-GAG scaffolds. Biomaterials.Feb.2005 26:433–441. [PubMed: 15275817]

3. Khademhosseini A, et al. Progress in Tissue Engineering. Scientific American. May.2009 300:64–71. [PubMed: 19438051]

4. Lazic T, Falanga V. Bioengineered Skin Constructs and Their Use in Wound Healing. Plastic andReconstructive Surgery. Jan.2011 127:75S–90S. [PubMed: 21200276]

5. Roberts SJ, et al. Clinical applications of musculoskeletal tissue engineering. British MedicalBulletin. Jun.2008 86:7–22. [PubMed: 18424445]

6. Atala A. Engineering organs. Current Opinion in Biotechnology. Oct.2009 20:575–592. [PubMed:19896823]

7. Macchiarini P, et al. Clinical transplantation of a tissue-engineered airway. Lancet. Dec.2008372:2023–2030. [PubMed: 19022496]

8. Gomes S, et al. Natural and genetically engineered proteins for tissue engineering. Progress inPolymer Science. Jan.2012 37:1–17. [PubMed: 22058578]

9. McAllister TN, et al. Cell-based therapeutics from an economic perspective: primed for acommercial success or a research sinkhole? Regenerative Medicine. Nov.2008 3:925–937.[PubMed: 18947313]

10. Peck M, et al. Tissue engineering by self-assembly. Materials Today. May.2011 14:218–224.

11. Fujihara Y, et al. Immunological response to tissue-engineered cartilage derived from auricularchondrocytes and a PLLA scaffold in transgenic mice. Biomaterials. Feb.2010 31:1227–1234.[PubMed: 19913296]

Zorlutuna et al.


NIH


NIH


NIH


12. Huang NF, Li S. Regulation of the Matrix Microenvironment for Stem Cell Engineering andRegenerative Medicine. Annals of Biomedical Engineering. Apr.2011 39:1201–1214. [PubMed:21424849]

13. Zamir E, Geiger B. Molecular complexity and dynamics of cell-matrix adhesions. Journal of CellScience. Oct.2001 114:3583–3590. [PubMed: 11707510]

14. Ifkovits JL, Burdick JA. Review: Photopolymerizable and degradable biomaterials for tissueengineering applications. Tissue Engineering. Oct.2007 13:2369–2385. [PubMed: 17658993]

15. Nichol JW, et al. Cell-laden microengineered gelatin methacrylate hydrogels. Biomaterials. Jul.2010 31:5536–5544. [PubMed: 20417964]

16. Hartgerink JD, et al. Peptide-amphiphile nanofibers: A versatile scaffold for the preparation ofself-assembling materials. Proceedings of the National Academy of Sciences. Apr.2002 99:5133–5138.

17. Bracalello A, et al. Design and Production of a Chimeric Resilin-, Elastin-, and Collagen-LikeEngineered Polypeptide. Biomacromolecules. Aug.2011 12:2957–2965. [PubMed: 21707089]

18. Gomes SC, et al. Antimicrobial functionalized genetically engineered spider silk. Biomaterials.Jun.2011 32:4255–4266. [PubMed: 21458065]

19. Numata K, et al. Silk-Based Nanocomplexes with Tumor-Homing Peptides for Tumor-SpecificGene Delivery. Macromolecular Bioscience. Jan.2012 12:75–82. [PubMed: 22052706]

20. Xia XX, et al. Tunable Self-Assembly of Genetically Engineered Silk-Elastin-like ProteinPolymers. Biomacromolecules. Nov.2011 12:3844–3850. [PubMed: 21955178]

21. Kitajima T, et al. Recombinant Human Gelatin Substitute With Photoreactive Properties for CellCulture and Tissue Engineering. Biotechnology and Bioengineering. Oct.2011 108:2468–2476.

22. Gillette BM, et al. Dynamic Hydrogels: Switching of 3D Microenvironments Using Two-Component Naturally Derived Extracellular Matrices. Advanced Materials. Feb.2010 22:686–691.[PubMed: 20217770]

23. Shin SR, et al. Carbon Nanotube Reinforced Hybrid Microgels as Scaffold Materials for CellEncapsulation. Acs Nano. Jan.2012 6:362–372. [PubMed: 22117858]

24. Cheng XG, et al. An electrochemical fabrication process for the assembly of anisotropicallyoriented collagen bundles. Biomaterials. Aug.2008 29:3278–3288. [PubMed: 18472155]

25. Builles N, et al. Use of magnetically oriented orthogonal collagen scaffolds for hemi-cornealreconstruction and regeneration. Biomaterials. Nov.2010 31:8313–8322. [PubMed: 20708260]

26. Tian H, et al. Biodegradable synthetic polymers: Preparation, functionalization and biomedicalapplication. Progress in Polymer Science. Feb.2012 37:237–280.

27. Sionkowska A. Current research on the blends of natural and synthetic polymers as newbiomaterials: Review. Progress in Polymer Science. Sep.2011 36:1254–1276.

28. Patterson J, Hubbell JA. Enhanced proteolytic degradation of molecularly engineered PEGhydrogels in response to MMP-1 and MMP-2. Biomaterials. Oct.2010 31:7836–7845. [PubMed:20667588]

29. Edlund U, et al. A Strategy for the Covalent Functionalization of Resorbable Polymers withHeparin and Osteoinductive Growth Factor. Biomacromolecules. Mar.2008 9:901–905. [PubMed:18247564]

30. Mahony O, et al. Silica-Gelatin Hybrids with Tailorable Degradation and Mechanical Propertiesfor Tissue Regeneration. Advanced Functional Materials. Nov.2010 20:3835–3845.

31. Moon JJ, et al. Biomimetic hydrogels with pro-angiogenic properties. Biomaterials. May.201031:3840–3847. [PubMed: 20185173]

32. Patterson J, Hubbell JA. SPARC-derived protease substrates to enhance the plasmin sensitivity ofmolecularly engineered PEG hydrogels. Biomaterials. Feb.2011 32:1301–1310. [PubMed:21040970]

33. Leslie-Barbick JE, et al. Micron-Scale Spatially Patterned, Covalently Immobilized VascularEndothelial Growth Factor on Hydrogels Accelerates Endothelial Tubulogenesis and IncreasesCellular Angiogenic Responses. Tissue Engineering Part A. Jan.2011 17:221–229. [PubMed:20712418]

Zorlutuna et al.


NIH


NIH


NIH


34. Saik JE, et al. Covalently immobilized platelet-derived growth factor-BB promotes angiogenesis inbiomimetic poly(ethylene glycol) hydrogels. Acta Biomaterialia. Jan.2011 7:133–143. [PubMed:20801242]

35. Kemppainen JM, Hollister SJ. Tailoring the mechanical properties of 3D-designed poly(glycerolsebacate) scaffolds for cartilage applications. Journal of Biomedical Materials Research Part A.Jul.2010 94A:9–18. [PubMed: 20091702]

36. Kenar H, et al. A 3D aligned microfibrous myocardial tissue construct cultured under transientperfusion. Biomaterials. Aug.2011 32:5320–5329. [PubMed: 21570112]

37. Wu W, et al. Artificial Niche Combining Elastomeric Substrate and Platelets Guides VascularDifferentiation of Bone Marrow Mononuclear Cells. Tissue Engineering Part A. Aug.201117:1979–1992. [PubMed: 21449713]

38. Crapo PM, Wang Y. Physiologic compliance in engineered small-diameter arterial constructsbased on an elastomeric substrate. Biomaterials. Mar.2010 31:1626–1635. [PubMed: 19962188]

39. Sant S, et al. Hybrid PGS-PCL microfibrous scaffolds with improved mechanical and biologicalproperties. Journal of Tissue Engineering and Regenerative Medicine. Apr.2011 5:283–291.[PubMed: 20669260]

40. Engelmayr GC, et al. Accordion-like honeycombs for tissue engineering of cardiac anisotropy.Nature Materials. Dec.2008 7:1003–1010.

41. Wu EC, et al. Self-Assembling Peptides as Cell-Interactive Scaffolds. Advanced FunctionalMaterials. Feb.2012 22:456–468.

42. Kieffer AE, et al. The N- and C-terminal fragments of ubiquitin are important for the antimicrobialactivities. Faseb Journal. Feb.2003 17:776–778. [PubMed: 12594174]

43. Loo Y, et al. From short peptides to nanofibers to macromolecular assemblies in biomedicine.Biotechnology Advances. May-Jun;2012 30:593–603. [PubMed: 22041166]

44. Mishra A, et al. Ultrasmall natural peptides self-assemble to strong temperature-resistant helicalfibers in scaffolds suitable for tissue engineering. Nano Today. Jun.2011 6:232–239.

45. Zhang SG. Fabrication of novel biomaterials through molecular self-assembly. NatureBiotechnology. Oct.2003 21:1171–1178.

46. Cho H, et al. Regulation of endothelial cell activation and angiogenesis by injectable peptidenanofibers. Acta Biomaterialia. Jan.2012 8:154–164. [PubMed: 21925628]

47. Galler KM, et al. A Customized Self-Assembling Peptide Hydrogel for Dental Pulp TissueEngineering. Tissue Engineering Part A. Jan.2012 18:176–184. [PubMed: 21827280]

48. Wang BC, et al. Functionalized self-assembling peptide nanofiber hydrogel as a scaffold for rabbitnucleus pulposus cells. Journal of Biomedical Materials Research Part A. Mar.2012 100A:646–653. [PubMed: 22213420]

49. Anderson JM, et al. Biphasic Peptide Amphiphile Nanomatrix Embedded with HydroxyapatiteNanoparticles for Stimulated Osteoinductive Response. Acs Nano. Dec.2011 5:9463–9479.[PubMed: 22077993]

50. Lenas P, et al. Modularity in developmental biology and artificial organs: A missing concept intissue engineering. Artificial Organs. Jun.2011 35:656–662. [PubMed: 21668829]

51. Davis NE, et al. Modular enzymatically crosslinked protein polymer hydrogels for in situ gelation.Biomaterials. Oct.2010 31:7288–7297. [PubMed: 20609472]

52. Serban MA, Prestwich GD. Modular extracellular matrices: Solutions for the puzzle. Methods.May.2008 45:93–98. [PubMed: 18442709]

53. Scott EA, et al. Modular scaffolds assembled around living cells using poly(ethylene glycol)microspheres with macroporation via a noncytotoxic porogen. Acta Biomaterialia. Jan.2010 6:29–38. [PubMed: 19607945]

54. Ehrbar M, et al. Enzymatic formation of modular cell-instructive fibrin analogs for tissueengineering. Biomaterials. Sep.2007 28:3856–3866. [PubMed: 17568666]

55. McGuigan AP, et al. Fabrication of cell-containing gel modules to assemble modular tissue-engineered constructs. Nature protocols. Mar.2006 1:2963–2969.

Zorlutuna et al.


NIH


NIH


NIH


56. Sosnik A, et al. Lactoyl-poloxamine/collagen matrix for cell-containing tissue engineeringmodules. Journal of Biomedical Materials Research - Part A. Aug.2008 86:339–353. [PubMed:17969022]

57. Leung BM, Sefton MV. A modular tissue engineering construct containing smooth muscle cellsand endothelial cells. Annals of Biomedical Engineering. Dec.2007 35:2039–2049. [PubMed:17882548]

58. McGuigan AP, Sefton MV. Vascularized organoid engineered by modular assembly enables bloodperfusion. Proceedings of the National Academy of Sciences of the United States of America.Aug.2006 103:11461–11466. [PubMed: 16864785]

59. Chamberlain MD, et al. Chimeric vessel tissue engineering driven by endothelialized modules inimmunosuppressed sprague-dawley rats. Tissue Engineering - Part A. Jan.2011 17:151–160.[PubMed: 20695789]

60. Leung BM, Sefton MV. A modular approach to cardiac tissue engineering. Tissue Engineering -Part A. Oct.2010 16:3207–3218. [PubMed: 20504074]

61. McGuigan AP, Sefton MV. The thrombogenicity of human umbilical vein endothelial cell seededcollagen modules. Biomaterials. Jun.2008 29:2453–2463. [PubMed: 18325586]

62. Khan OF, Sefton MV. Endothelial cell behaviour within a microfluidic mimic of the flow channelsof a modular tissue engineered construct. Biomedical Microdevices. Jan.2011 13:69–87. [PubMed:20842530]

63. Du Y, et al. Surface-directed assembly of cell-laden microgels. Biotechnology and Bioengineering.Feb.2010 105:655–62. [PubMed: 19777588]

64. Du Y, et al. Sequential assembly of cell-laden hydrogel constructs to engineer vascular-likemicrochannels. Biotechnology and Bioengineering. Jul.2011 108:1693–1703. [PubMed:21337336]

65. Du Y, et al. Convection-driven generation of long-range material gradients. Biomaterials. Mar.2010 31:2686–94. [PubMed: 20035990]

66. Du Y, et al. Directed assembly of cell-laden microgels for fabrication of 3D tissue constructs.Proceedings of the National Academy of Sciences of the United States of America. Jul.2008105:9522–7. [PubMed: 18599452]

67. Du Y, et al. Method of Bottom-Up Directed Assembly of Cell-Laden Microgels. Cellular andMolecular Bioengineering. Feb.2008 1:157–162. [PubMed: 19953195]

68. Zamanian B, et al. Interface-directed self-assembly of cell-laden microgels. Small. Apr.20106:937–44. [PubMed: 20358531]

69. Yamaguchi H, et al. Photoswitchable gel assembly based on molecular recognition. NatureCommunications. Jan.2012 3

70. Harada A, et al. Macroscopic self-assembly through molecular recognition. Nature Chemistry.2011; 3:34–37.

71. Gartner ZJ, Bertozzi CR. Programmed assembly of 3-dimensional microtissues with definedcellular connectivity. Proceedings of the National Academy of Sciences of the United States ofAmerica. Mar.2009 106:4606–4610. [PubMed: 19273855]

72. Jewett JC, Bertozzi CR. Cu-free click cycloaddition reactions in chemical biology. ChemicalSociety Reviews. Jan.2010 39:1272–1279. [PubMed: 20349533]

73. Selden NS, et al. Chemically programmed cell adhesion with membrane-anchoredoligonucleotides. Journal of the American Chemical Society. 2012; 134:765–768. [PubMed:22176556]

74. Dutta D, et al. Synthetic chemoselective rewiring of cell surfaces: Generation of three-dimensionaltissue structures. Journal of the American Chemical Society. 2011; 133:8704–8713. [PubMed:21561150]

75. Bratt-Leal AM, et al. Magnetic manipulation and spatial patterning of multi-cellular stem cellaggregates. Integrative Biology. Mar.2011 3:1224–1232. [PubMed: 22076329]

76. Jiao T, et al. Measurements of the Effects of Decellularization on Viscoelastic Properties ofTissues in Ovine, Baboon, and Human Heart Valves. Tissue Engineering Part A. Feb.201218:423–431. [PubMed: 21919799]

Zorlutuna et al.


NIH


NIH


NIH


77. Remlinger NT, et al. Hydrated xenogeneic decellularized tracheal matrix as a scaffold for trachealreconstruction. Biomaterials. May.2010 31:3520–3526. [PubMed: 20144481]

78. Vorotnikova E, et al. Extracellular matrix-derived products modulate endothelial and progenitorcell migration and proliferation in vitro and stimulate regenerative healing in vivo. MatrixBiology. Oct.2010 29:690–700. [PubMed: 20797438]

79. Crapo PM, et al. An overview of tissue and whole organ decellularization processes. Biomaterials.Apr.2011 32:3233–3243. [PubMed: 21296410]

80. Badylak SF, et al. Esophageal Preservation in Five Male Patients After Endoscopic Inner-LayerCircumferential Resection in the Setting of Superficial Cancer: A Regenerative MedicineApproach with a Biologic Scaffold. Tissue Engineering Part A. Jun.2011 17:1643–1650.[PubMed: 21306292]

81. Keane TJ, et al. Consequences of ineffective decellularization of biologic scaffolds on the hostresponse. Biomaterials. Feb.2012 33:1771–1781. [PubMed: 22137126]

82. Gilbert TW, et al. A quantitative method for evaluating the degradation of biologic scaffoldmaterials. Biomaterials. Jan.2007 28:147–50. [PubMed: 16949150]

83. L'Heureux N, et al. A completely biological tissue-engineered human blood vessel. Faseb Journal.Jan.1998 12:47–56. [PubMed: 9438410]

84. Haraguchi Y, et al. Concise Review: Cell Therapy and Tissue Engineering for CardiovascularDisease. Stem Cells Translational Medicine. Feb.2012 1:136–141. [PubMed: 23197760]

85. Napolitano AP, et al. Scaffold-free three-dimensional cell culture utilizing micromoldednonadhesive hydrogels. Biotechniques. Oct.2007 43:496–500.

86. Karp JM, et al. Controlling size, shape and homogeneity of embryoid bodies using poly(ethyleneglycol) microwells. Lab Chip. Jun.2007 7:786–94. [PubMed: 17538722]

87. Hwang YS, et al. Microwell-mediated control of embryoid body size regulates embryonic stem cellfate via differential expression of WNT5a and WNT11. Proc Natl Acad Sci U S A. Oct.2009106:16978–83. [PubMed: 19805103]

88. Choi YS, et al. Mechanical derivation of functional myotubes from adipose-derived stem cells.Biomaterials. Mar.2012 33:2482–2491. [PubMed: 22197570]

89. Tekin H, et al. Stimuli-responsive microwells for formation and retrieval of cell aggregates. Lab ona Chip - Miniaturisation for Chemistry and Biology. Oct.2010 10:2411–2418.

90. Khademhosseini A, Langer R. Microengineered hydrogels for tissue engineering. Biomaterials.Dec.2007 28:5087–92. [PubMed: 17707502]

91. Tekin H, et al. Responsive microgrooves for the formation of harvestable tissue constructs.Langmuir. May.2011 27:5671–9. [PubMed: 21449596]

92. Gallego-Perez D, et al. High throughput assembly of spatially controlled 3D cell clusters on amicro/nanoplatform. Lab on a Chip - Miniaturisation for Chemistry and Biology. Mar.201010:775–782.

93. Livoti CM, Morgan JR. Self-Assembly and Tissue Fusion of Toroid-Shaped Minimal BuildingUnits. Tissue Engineering Part A. Jun.2010 16:2051–2061. [PubMed: 20109063]

94. Wong SF, et al. Concave microwell based size-controllable hepatosphere as a three-dimensionalliver tissue model. Biomaterials. Nov.2011 32:8087–8096. [PubMed: 21813175]

95. Akins RE, et al. Three-dimensional culture alters primary cardiac cell phenotype. TissueEngineering - Part A. Feb.2010 16:629–641. [PubMed: 20001738]

96. Qi H, et al. Patterned differentiation of individual embryoid bodies in spatially organized 3Dhybrid microgels. Advanced Materials. Dec.2010 22:5276–5281. [PubMed: 20941801]

97. Spelke DP, et al. Methods for embryoid body formation: the microwell approach. Methods inmolecular biology (Clifton, N.J.). 2011; 690:151–162.

98. Bratt-Leal AM, et al. Engineering the embryoid body microenvironment to direct embryonic stemcell differentiation. Biotechnology Progress. Jan-Feb;2009 25:43–51. [PubMed: 19198003]

99. Gerecht S, et al. Hyaluronic acid hydrogel for controlled self-renewal and differentiation of humanembryonic stem cells. Proceedings of the National Academy of Sciences of the United States ofAmerica. Jul.2007 104:11298–303. [PubMed: 17581871]

Zorlutuna et al.


NIH


NIH


NIH


100. Moeller H-C, et al. A microwell array system for stem cell culture. Biomaterials. Feb.200829:752–763. [PubMed: 18001830]

101. Choi YY, et al. Controlled-size embryoid body formation in concave microwell arrays.Biomaterials. May.2010 31:4296–4303. [PubMed: 20206991]

102. Selimovic S, et al. Microfabricated polyester conical microwells for cell culture applications. Labon a Chip - Miniaturisation for Chemistry and Biology. Nov.2011 11:2325–2332.

103. Bratt-Leal AM, et al. Incorporation of biomaterials in multicellular aggregates modulatespluripotent stem cell differentiation. Biomaterials. Jan.2011 32:48–56. [PubMed: 20864164]

104. Lin JB, et al. Thermo-responsive poly(N-isopropylacrylamide) grafted onto microtexturedpoly(dimethylsiloxane) for aligned cell sheet engineering. Colloids and Surfaces B:Biointerfaces. Nov.2012 99:108–115.

105. Elloumi-Hannachi I, et al. Cell sheet engineering: a unique nanotechnology for scaffold-freetissue reconstruction with clinical applications in regenerative medicine. Journal of InternalMedicine. Jan.2010 267:54–70. [PubMed: 20059644]

106. Isenberg BC, et al. Micropatterned cell sheets with defined cell and extracellular matrixorientation exhibit anisotropic mechanical properties. Journal of Biomechanics. Mar.201245:756–761. [PubMed: 22177672]

107. Sekine H, et al. Myocardial tissue engineering: toward a bioartificial pump. Cell and TissueResearch. Mar.2012 347:775–782. [PubMed: 22095463]

108. Haraguchi Y, et al. Regenerative therapies using cell sheet-based tissue engineering for cardiacdisease. Cardiology Research and Practice. 2011; 1

109. Sekine H, et al. Cardiac cell sheet transplantation improves damaged heart function via superiorcell survival in comparison with dissociated cell injection. Tissue Engineering - Part A. Nov.2011 17:2973–2980. [PubMed: 21875331]

110. Matsuura K, et al. Creation of mouse embryonic stem cell-derived cardiac cell sheets.Biomaterials. Oct.2011 32:7355–7362. [PubMed: 21807408]

111. Huang C-C, et al. A strategy for fabrication of a three-dimensional tissue construct containinguniformly distributed embryoid body-derived cells as a cardiac patch. Biomaterials. Aug.201031:6218–6227. [PubMed: 20537702]

112. Ohashi K, et al. Engineering functional two- and three-dimensional liver systems in vivo usinghepatic tissue sheets. Nature Medicine. Jun.2007 13:880–885.

113. Kim K, et al. Preserved liver-specific functions of hepatocytes in 3D co-culture with endothelialcell sheets. Biomaterials. 2012; 33:1406–1413. [PubMed: 22118777]

114. Wei FL, et al. Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissueregeneration by elevating telomerase activity. Journal of Cellular Physiology. Sep.2011227:3216–3224. [PubMed: 22105792]

115. Solorio LD, et al. Engineered cartilage via self-assembled hMSC sheets with incorporatedbiodegradable gelatin microspheres releasing transforming growth factor-β1. Journal ofControlled Release. Mar.2012 158:224–232. [PubMed: 22100386]

116. Zahn R, et al. Ion-induced cell sheet detachment from standard cell culture surfaces coated withpolyelectrolytes. Biomaterials. Apr.2012 33:3421–3427. [PubMed: 22300744]

117. Thomson JA, et al. Embryonic stem cell lines derived from human blastocysts. Science. Nov.1998 282:1145–7. [PubMed: 9804556]

118. Watt FM, Hogan BL. Out of Eden: stem cells and their niches. Science. Feb.2000 287:1427–30.[PubMed: 10688781]

119. Mummery C, et al. Differentiation of human embryonic stem cells to cardiomyocytes: role ofcoculture with visceral endoderm-like cells. Circulation. Jun.2003 107:2733–40. [PubMed:12742992]

120. Kehat I, et al. Human embryonic stem cells can differentiate into myocytes with structural andfunctional properties of cardiomyocytes. Journal of Clinical Investigation. Aug.2001 108:407–14. [PubMed: 11489934]

121. Kehat I, et al. High-resolution electrophysiological assessment of human embryonic stem cell-derived cardiomyocytes: a novel in vitro model for the study of conduction. CirculationResearch. Oct.2002 91:659–61. [PubMed: 12386141]

Zorlutuna et al.


NIH


NIH


NIH


122. Levenberg S, et al. Endothelial cells derived from human embryonic stem cells. Proceedings ofthe National Academy of Sciences of the United States of America. Apr.2002 99:4391–6.[PubMed: 11917100]

123. Hamazaki T, et al. Hepatic maturation in differentiating embryonic stem cells in vitro. FEBSLetters. May.2001 497:15–9. [PubMed: 11376655]

124. Ishii T, et al. In vitro differentiation and maturation of mouse embryonic stem cells intohepatocytes. Experimental Cell Research. Jul.2005

125. Lavon N, et al. Differentiation and isolation of hepatic-like cells from human embryonic stemcells. Differentiation. Jun.2004 72:230–8. [PubMed: 15270779]

126. Moritoh Y, et al. Analysis of insulin-producing cells during in vitro differentiation from feeder-free embryonic stem cells. Diabetes. May.2003 52:1163–8. [PubMed: 12716747]

127. Bielby RC, et al. In vitro differentiation and in vivo mineralization of osteogenic cells derivedfrom human embryonic stem cells. Tissue Engineering. Sep-Oct;2004 10:1518–25. [PubMed:15588411]

128. Karp JM, et al. Cultivation of human embryonic stem cells without the embryoid body stepenhances osteogenesis in vitro. Stem Cells. Apr.2006 24:835–843. [PubMed: 16253980]

129. Kim JH, et al. Dopamine neurons derived from embryonic stem cells function in an animal modelof Parkinson's disease. Nature. Jul.2002 418:50–6. [PubMed: 12077607]

130. Bjorklund LM, et al. Embryonic stem cells develop into functional dopaminergic neurons aftertransplantation in a Parkinson rat model. Proceedings of the National Academy of Sciences of theUnited States of America. Feb.2002 99:2344–9. [PubMed: 11782534]

131. Zhang SC, et al. In vitro differentiation of transplantable neural precursors from humanembryonic stem cells. Nature Biotechnology. Dec.2001 19:1129–33.

132. Reubinoff BE, et al. Neural progenitors from human embryonic stem cells. Nature Biotechnology.Dec.2001 19:1134–40.

133. Schuldiner M, et al. Induced neuronal differentiation of human embryonic stem cells. BrainResearch. Sep.2001 913:201–5. [PubMed: 11549388]

134. Manzar N, et al. The Ethical Dilemma of Embryonic Stem Cell Research. Science andEngineering Ethics. 2011:1–10.

135. Bilic J, Izpisua Belmonte JC. Concise review: Induced pluripotent stem cells versus embryonicstem cells: Close enough or yet too far apart? Stem Cells. Jan.2012 30:33–41. [PubMed:22213481]

136. Graf T, Enver T. Forcing cells to change lineages. Nature. Dec.2009 462:587–594. [PubMed:19956253]

137. Fluri DA, et al. Derivation, expansion and differentiation of induced pluripotent stem cells incontinuous suspension cultures. Nature Methods. Mar.2012

138. Seki T, et al. Generation of induced pluripotent stem cells from a small amount of humanperipheral blood using a combination of activated T cells and Sendai virus. Nature Protocols.Mar.2012 7:718–728.

139. Nagamoto Y, et al. The promotion of hepatic maturation of human pluripotent stem cells in 3Dco-culture using type I collagen and Swiss 3T3 cell sheets. Biomaterials. Jun.2012 33:4526–4534. [PubMed: 22445253]

140. Josowitz R, et al. Induced pluripotent stem cell-derived cardiomyocytes as models for geneticcardiovascular disorders. Current Opinion in Cardiology. May.2011 26:223–229. [PubMed:21451408]

141. Tulloch NL, et al. Growth of engineered human myocardium with mechanical loading andvascular coculture. Circulation Research. May.2011 109:47–59. [PubMed: 21597009]

142. Hussein SM, et al. Copy number variation and selection during reprogramming to pluripotency.Nature. Mar.2011 471:58–62. [PubMed: 21368824]

143. Gore A, et al. Somatic coding mutations in human induced pluripotent stem cells. Nature. Mar.2011 471:63–67. [PubMed: 21368825]

144. Pera MF. Stem cells: The dark side of induced pluripotency. Nature. Mar.2011 471:46–47.[PubMed: 21368819]

Zorlutuna et al.


NIH


NIH


NIH


145. Burdick JA, Vunjak-Novakovic G. Engineered microenvironments for controlled stem celldifferentiation. Tissue Engineering - Part A. Feb.2009 15:205–219. [PubMed: 18694293]

146. Dado D, et al. Mechanical control of stem cell differentiation. Regenerative Medicine. Jan.20127:101–116. [PubMed: 22168501]

147. Hudalla GA, Murphy WL. Biomaterials that regulate growth factor activity via bioinspiredinteractions. Advanced Functional Materials. May.2011 21:1754–1768. [PubMed: 21921999]

148. Toh WS, et al. Modulation of mesenchymal stem cell chondrogenesis in a tunable hyaluronic acidhydrogel microenvironment. Biomaterials. May.2012 33:3835–3845. [PubMed: 22369963]

149. Sharma RI, Snedeker JG. Paracrine interactions between mesenchymal stem cells affect substratedriven differentiation toward tendon and bone phenotypes. PLoS ONE. Feb.2012 7

150. Zhang G, et al. Vascular differentiation of bone marrow stem cells is directed by a tunable three-dimensional matrix. Acta Biomaterialia. Sep.2010 6:3395–3403. [PubMed: 20302976]

151. Seidlits SK, et al. The effects of hyaluronic acid hydrogels with tunable mechanical properties onneural progenitor cell differentiation. Biomaterials. May.2010 31:3930–3940. [PubMed:20171731]

152. Minato A, et al. Cardiac differentiation of embryonic stem cells by substrate immobilization ofinsulin-like growth factor binding protein 4 with elastin-like polypeptides. Biomaterials. Jan.2012 33:515–523. [PubMed: 22018385]

153. Wylie RG, et al. Spatially controlled simultaneous patterning of multiple growth factors in three-dimensional hydrogels. Nature Materials. Oct.2011 10:799–806.

154. Koffas M, et al. Metabolic engineering. Annual Review of Biomedical Engineering. Aug.19991:535–557.

155. Graf A, et al. Yeast systems biotechnology for the production of heterologous proteins. FemsYeast Research. May.2009 9:335–348. [PubMed: 19341379]

156. Ruffing A, Chen RR. Metabolic engineering of microbes for oligosaccharide and polysaccharidesynthesis. Microbial Cell Factories. Jul.2006 5

157. Iwase A, et al. Manipulation of plant metabolic pathways by transcription factors. PlantBiotechnology. Mar.2009 26:29–38.

158. Bailey JE, et al. Inverse metabolic engineering: A strategy for directed genetic engineering ofuseful phenotypes. Biotechnology and Bioengineering. Oct.1996 52:109–121. [PubMed:18629857]

159. Tyo KE, et al. Expanding the metabolic engineering toolbox: more options to engineer cells.Trends in Biotechnology. Mar.2007 25:132–137. [PubMed: 17254656]

160. Godia F, Cairo JJ. Metabolic engineering of animal cells. Bioprocess and BiosystemsEngineering. Jan.2002 24:289–298.

161. Baik JY, et al. Metabolic engineering of Chinese hamster ovary cells: Towards a bioengineeredheparin. Metabolic engineering. Mar.2012 14:81–90. [PubMed: 22326251]

162. Pouilly S, et al. Metabolic glycoengineering through the mammalian GalNAc salvage pathway.Febs Journal. Feb.2012 279:586–598. [PubMed: 22151230]

163. Du J, Yarema KJ. Carbohydrate engineered cells for regenerative medicine. Adv Drug DeliveryReviews. Jun.2010 62:671–682.

164. Seth G, et al. Hu WSST. Engineering cells for cell culture bioprocessing - Physiologicalfundamentals. Cell Culture Engineering. 2006; 101:119–164.

165. Shanmugarajan TS, et al. Growth Factors and Signaling Pathways in the ChondrogenicDifferentiation of Mesenchymal Stem Cells. Tissue Engineering and Regenerative Medicine.Jun.2011 8:292–299.

166. Osborne R, et al. Understanding Metabolic Pathways for Skin Anti-Aging. Journal of Drugs inDermatology. Jul.2009 8:S4–S7. [PubMed: 19623777]

167. Hofmann A, et al. Osteoblasts - Cellular and molecular regulatory mechanisms in fracturehealing. Orthopade. Nov.2009 38:1009–1019. [PubMed: 19826779]

168. Kurinna S, Barton MC. Cascades of transcription regulation during liver regeneration.International Journal of Biochemistry & Cell Biology. Feb.2011 43:189–197. [PubMed:20307684]

Zorlutuna et al.


NIH


NIH


NIH


169. Majors BS, et al. Links between metabolism and apoptosis in mammalian cells: Applications foranti-apoptosis engineering. Metabolic Engineering. Jul.2007 9:317–326. [PubMed: 17611135]

170. Menendez L, et al. Wnt signaling and a Smad pathway blockade direct the differentiation ofhuman pluripotent stem cells to multipotent neural crest cells. Proceedings of the NationalAcademy of Sciences of the United States of America. Nov.2011 108:19240–19245. [PubMed:22084120]

171. Sackstein R. Glycoengineering of HCELL, the Human Bone Marrow Homing Receptor: SweetlyProgramming Cell Migration. Annals of Biomedical Engineering. Apr.2012 40:766–76.[PubMed: 22068886]

172. Derda R, et al. Multizone Paper Platform for 3D Cell Cultures. Plos One. May.2011 6

173. Fox S, et al. High-Throughput Screening: Update on Practices and Success. Journal ofBiomolecular Screening. Oct.2006 11:864–869. [PubMed: 16973922]

174. Xu Y, et al. A Microfluidic Hydrogel Capable of Cell Preservation without Perfusion Cultureunder Cell-Based Assay Conditions. Advanced Materials. Jul.2010 22:3017–3021. [PubMed:20503209]

175. Lan SF, Starly B. Alginate based 3D hydrogels as an in vitro co-culture model platform for thetoxicity screening of new chemical entities. Toxicology and Applied Pharmacology. Oct.2011256:62–72. [PubMed: 21839104]

176. Shultz LD, et al. Humanized mice in translational biomedical research. Nature ReviewsImmunology. Feb.2007 7:118–130.

177. Giese C, et al. Immunological substance testing on human lymphatic micro-organoids in vitro.Journal of Biotechnology. Jul.2010 148:38–45. [PubMed: 20416346]

178. Vrana NE, et al. Development of a Reconstructed Cornea from Collagen–Chondroitin SulfateFoams and Human Cell Cultures. Investigative Ophthalmology & Visual Science. Dec.200849:5325–5331. [PubMed: 18708614]

179. Sung JH, et al. Microscale 3-D hydrogel scaffold for biomimetic gastrointestinal (GI) tract model.Lab on a Chip. Nov.2011 11:389–392. [PubMed: 21157619]

180. Huh D, et al. Reconstituting Organ-Level Lung Functions on a Chip. Science. Jun.2010328:1662–1668. [PubMed: 20576885]

181. Vozzi F, et al. Connected Culture of Murine Hepatocytes and HUVEC in a MulticompartmentalBioreactor. Tissue Engineering Part A. Jun.2009 15:1291–1299. [PubMed: 18837649]

182. Ingber D. ‘Organ-on-a-chip’ technology: on trial. Chemistry & Industry. Jul.2011 :18–20.

183. Esch, MB., et al. The Role of Body-on-a-Chip Devices in Drug and Toxicity Studies. In:Yarmush, ML., et al., editors. Annual Review of Biomedical Engineering, Vol 13. Vol. 13.Annual Reviews; Palo Alto: 2011. p. 55-72.

184. Zhang C, et al. Towards a human-on-chip: Culturing multiple cell types on a chip withcompartmentalized microenvironments. Lab on a Chip. Sep.2009 9:3185–3192. [PubMed:19865724]

185. Chia SM, et al. TGF-beta 1 regulation in hepatocyte-NIH3T3 co-culture is important for theenhanced hepatocyte function in 3D microenvironment. Biotechnology and Bioengineering. Mar.2005 89:565–573. [PubMed: 15669090]

186. Kim SB, et al. A cell-based biosensor for real-time detection of cardiotoxicity using lensfreeimaging. Lab on a Chip. Nov.2011 11:1801–1807. [PubMed: 21483937]

187. Enejder A, et al. Periasamy A, et al.CARS and SHG microscopy of artificial, bioengineeredtissues. Multiphoton Microscopy in the Biomedical Sciences X. 2010; 7569

188. Brackmann C, et al. Visualization of the Cellulose Biosynthesis and Cell Integration intoCellulose Scaffolds. Biomacromolecules. Mar.2010 11:542–548. [PubMed: 20158282]

189. Choi JR, et al. Investigation of portable in situ fluorescence optical detection for microfluidic 3Dcell culture assays. Optics Letters. May.2010 35:1374–1376. [PubMed: 20436574]

190. Tatosian DA, Shuler ML. A Novel System for Evaluation of Drug Mixtures for Potential Efficacyin Treating Multidrug Resistant Cancers. Biotechnology and Bioengineering. May.2009103:187–198. [PubMed: 19137589]

Zorlutuna et al.


NIH


NIH


NIH


191. Götz C, et al. Xenobiotic Metabolism Capacities Of Human Skin In Comparison to a 3D-epidermis model and keratinocyte-based cell culture as In vitro alternatives for chemical testing :phase ii enzymes. Experimental Dermatology. May.2012 :358–363. [PubMed: 22509833]

192. Jong Bin K. Three-dimensional tissue culture models in cancer biology. Seminars in CancerBiology. Oct.2005 15:365–377. [PubMed: 15975824]

193. Ingber DE, et al. Tissue engineering and developmental biology: Going biomimetic. TissueEngineering. Dec.2006 12:3265–3283. [PubMed: 17518669]

194. Lee GY, et al. Three-dimensional culture models of normal and malignant breast epithelial cells.Nature Methods. Mar.2007 4:359–365. [PubMed: 17396127]

195. Mani SA, et al. The epithelial-mesenchymal transition generates cells with properties of stemcells. Cell. May.2008 133:704–715. [PubMed: 18485877]

196. Sewell-Loftin MK, et al. EMT-Inducing Biomaterials for Heart Valve Engineering: Taking Cuesfrom Developmental Biology. Journal of Cardiovascular Translational Research. Oct.20114:658–671. [PubMed: 21751069]

197. Han B, et al. A cryoinjury model using engineered tissue equivalents for cryosurgicalapplications. Annals of Biomedical Engineering. Jul.2005 33:972–982. [PubMed: 16060538]

198. Ueda J, et al. Large Effective-Strain Piezoelectric Actuators Using Nested Cellular ArchitectureWith Exponential Strain Amplification Mechanisms. Ieee-Asme Transactions on Mechatronics.Oct.2010 15:770–782.

199. Nagai M, et al. Chemical control of Vorticella bioactuator using microfluidics. Lab on a Chip.Oct.2010 10:1574–1578. [PubMed: 20449516]

200. Akiyama Y, et al. Long-term and room temperature operable bioactuator powered by insectdorsal vessel tissue. Lab on a Chip. Aug.2009 9:140–144. [PubMed: 19209346]

201. Xi J, et al. Self-assembled microdevices driven by muscle. Nature Materials. Jan.2005 4:180–184.

202. Yamamoto Y, et al. Functional Evaluation of Artificial Skeletal Muscle Tissue ConstructsFabricated by a Magnetic Force-Based Tissue Engineering Technique. Tissue Engineering PartA. Jan.2011 17:107–114. [PubMed: 20672996]

203. Horiguchi H, et al. Fabrication and Evaluation of Reconstructed Cardiac Tissue and ItsApplication to Bio-actuated Microdevices. Ieee Transactions on Nanobioscience. Dec.20098:349–355. [PubMed: 20142148]

204. Ferrandez JM, et al. Human Neuroblastoma Cultures for Biorobotics. Sep.2011 :6672–6675.

205. Kubon M, et al. A Microsensor System to Probe Physiological Environments and TissueResponse. 2010 Ieee Sensors. 2010:2607–2611.

206. Brackmann C, et al. Non-linear microscopy of smooth muscle cells in artificial extracellularmatrices made of cellulose. Journal of Biophotonics. May.2012 5:404–414. [PubMed: 22461222]

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Figure 1.Control of cell spreading by reversibly crosslinkable ECM components, by selectivelycrosslinking, uncrosslinking and re-crosslinking the alginate component within the collagenhydrogel, cell shape can be controlled in 3D. Cells were labeled for actin fibers withAlexiFluor488-Phalloidin (Adapted from [22]).

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Figure 2.Hexamer polypeptides with a hydrophilic head and a hydrophobic tail can self- assembleinto millimeter scale fibers. A) Schematic model of the hexamer polypeptide sequences(LIVAGD and AIVAGD) that assemble into fibers via α-helical pairing. B) Fiber mats andsingle fibers obtained from the self-assembling polypeptides, these fibers can supportseveral cell types such as mesenchymal stem cells and epithelial cells (Adapted from [44]).

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Figure 3.A) Schematics of the modular construct design and fabrication. HepG2 cell encapsulatedcylindrical collagen gels were seeded with human umbilical-vein endothelial cells(HUVECs). After HUVECs completely covered the gel surface, which takes usually 2 to 3days, the HUVEC-seeded cylinders can be assembled into a larger structure to form themodular construct that can be used to perfuse supply nutrients to the cells through formednetwork of interconnected channels. B) Light microscopy images of collagen–HepG2module prior to HUVEC seeding. C) On Day 7 of the culture period, HUVECs on thesurface of the modules was stained for VE-cadherin and imaged using confocal microscopyto show the confluent layer of HUVECs on the surface of the modules. D) A flow circuitwas used to perfuse PBS or media the modular construct. E) After media perfusion of 7days, collagen–HepG2–HUVEC modules were retrieved and imaged using confocalmicroscopy (Adapted from [58]).

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Figure 4.Macroscale assembly of gel modules through selective molecular recognition of acrylamidegels modified with cyclodextrin or hydrocarbon groups. A) Selective assembly of α-CD-gel(blue) and n-Bu-gel (yellow) upon shaking in water in the presence of t-Bu-gel (dark green).B) Selective assembly of β-CD-gel (red) and t-Bu-gel (dark green) upon shaking in water inthe presence of n-Bu-gel (yellow). C) Selective assembly of α-CD-gel/n-Bu-gel and β-CD-gel/t-Bu-gel upon shaking in water in the presence of all gel types (Adapted from [70]).

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Figure 5.Fabrication of temperature responsive substrates for generating cell aggregates. A-B)PNIPAAm microwells were fabricated by soft lithography, then cells were seeded andretrieved after aggregate formation. Phase contrast and fluorescent microscopy images of theaggregates released from C) PNIPAAm compared to D) PEG microwells (Adapted from[89]).

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Figure 6.A) Schematics of the simultaneous protein immobilization using 2 photon laser scanningmethod. A femtosecond laser was used to immobilize maleimide-barnase, represented withthe black circle, followed by a wash step to remove unbound maleimide-barnase. Then,maleimide-streptavidin was immobilized, represented by orange square, and again followedby a wash step. After this step, a protein of interest which has been fused to barstar andbiotin was introduced. They will specifically bind to barnase and streptavidin, respectively.B and C) Confocal microscopy images of simultaneous patterning of biotin–CNTF (red) andbarstar–SHH (green) (Scale bar: 100 μm) (Adapted from [153]).

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Figure 7.Non-linear microscopy techniques SHG and CARS allow simultaneous, real-timemonitoring of cell-fibrous scaffold (bacterial cellulose) interactions. A) Interactions ofvascular smooth muscle cells with nanofibrillar bacterial cellulose was monitored at specifictime points to quantify cell proliferation, migration and ECM secretion B) Secreted collagenfibers can be distinguished from the cellulose fibers due to the substantial difference in theiraverage fiber size (A collagen fiber is indicated with an arrow around vascular smoothmuscle cells) (Adapted from [206]).

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